P131 is a protein that inhibits degradation of synthetic peptides and large protein substrates by the 20S proteasome (Ma et al., Biochim. Biophys. Acta 1119 (1992), 303-311). We have cloned and sequenced the human gene for P131. The deduced primary structure of P131, 271 amino acids, has a molecular weight of 29,792 in accord with that estimated by SDS-PAGE for the protein purified from bovine blood cells. P131 is not similar to any previously reported protein and has a proline-rich carboxyterminal half (26% proline residues). Recombinant P131 has been expressed in E. coli, purified to homogeneity, and found to display inhibitory properties similar to those of bovine P131. Native P131, a 60 kDa dimer as determined by gel filtration, inhibits proteasome activity by directly binding to the 20S proteasome in a 2:1 molar ratio. A proteasome-P131 complex has been isolated by glycerol density gradient centrifugation. We have constructed deletion mutants which show that the proline-rich carboxyterminus of p131 is responsible for inhibitory function. Circular-dichroism revealed that the proline-rich region has a random coil conformation suggesting that both the sequence of P131 and an extended random coil structure are important for inhibition. This lead to the synthesis of small peptide fragments of p131, and we have demonstrated that the inhibitory activity is localized to a small region within the proline-rich carboxyterminus. In addition, we have shown that P131 can inhibit the proteasome complexed to PA28, a proteasome activator believed to function in the immune response. P131 did not, however, inhibit the proteasome complexed to PA700, a multisubunit regulatory protein involved in ubiquitin-dependent protein degradation. These data indicate that there is a hierarchy among regulators for the control of proteasome function.